Changes in Structural-Mechanical Properties and Degradability of Collagen during Aging-associated Modifications

During aging, changes occur in the collagen network that contribute to various pathological phenotypes in the skeletal, vascular, and pulmonary systems. The aim of this study was to investigate the consequences of age-related modifications on the mechanical stability and in vitro proteolytic degradation of type I collagen. Analyzing mouse tail and bovine bone collagen, we found that collagen at both fibril and fiber levels varies in rigidity and Young''s modulus due to different physiological changes, which correlate with changes in cathepsin K (CatK)-mediated degradation. A decreased susceptibility to CatK-mediated hydrolysis of fibrillar collagen was observed following mineralization and advanced glycation end product-associated modification. However, aging of bone increased CatK-mediated osteoclastic resorption by ∼27%, and negligible resorption was observed when osteoclasts were cultured on mineral-deficient bone. We observed significant differences in the excavations generated by osteoclasts and C-terminal telopeptide release during bone resorption under distinct conditions. Our data indicate that modification of collagen compromises its biomechanical integrity and affects CatK-mediated degradation both in bone and tissue, thus contributing to our understanding of extracellular matrix aging.     

Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclasts

Estrogen deficiency arising with the menopause promotes marked acceleration of bone resorption, which can be restored by hormone replacement therapy. The inhibitory effects of estrogen seem to involve indirect cytokine- mediated effects via supporting bone marrow cells, but direct estrogen-receptor mediated effects on the bone-resorbing osteoclasts have also been proposed. Little information is available on whether estrogens modulate human osteoclastogenesis or merely inhibit the functional activity of osteoclasts. To clarify whether estrogens directly modulate osteoclastic activities human CD14+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1-10 nM 17beta-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17beta-estradiol inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17beta-estradiol (0.001-10 nM) to mature osteoclasts did not affect bone resorption or TRACP activity. We investigated expression of the estrogen receptors, using immunocytochemistry and Western blotting. We found that ER-alpha is expressed in osteoclast precursors, whereas ER- beta is expressed at all stages, indicating that the inhibitory effect of estrogen on osteoclastogenesis is mediated by ER-alpha for the major part. In conclusion, these results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts.     

Breast cancer cell line MDA-231 stimulates osteoclastogenesis and bone resorption in human osteoclasts

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. In the present study we utilized a well-known human breast cancer cell line (MDA-231) in order to assess its capability to influence osteoclastogenesis in human bone marrow cultures and bone resorption in fully differentiated osteoclasts. We demonstrated that conditioned medium (CM) harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures. Bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of MDA-231 CM. Moreover, while MDA-231 by themselves did not produce IL-6 tumor cell, CM increased the secretion of IL-6 by primary human osteoclasts and GCT cell lines compared to untreated controls. These data suggest that MDA-231 produce osteoclastic activating factor(s) that increase both osteoclast formation in bone marrow culture and bone resorption activity by mature cells. Moreover, breast cancer cells stimulate IL-6 secretion by osteoclasts that is one of the factors known to supports osteoclastogenesis.     

Dexamethasone inhibits bone resorption by indirectly inducing apoptosis of the bone-resorbing osteoclasts via the action of osteoblastic cells

Although glucocorticoids (GCs) are physiologically essentialfor bone metabolism, it is generally accepted that high dosesof GCs cause bone loss through a combination of decreased boneformation and increased bone resorption. However, the actionof GCs on mature osteoclasts remains contradictory. In thisstudy, we have examined the effect of GCs on osteoclasticbone-resorbing activity and osteoclast apoptosis, by using twodifferent cell types, rabbit unfractionated bone cells andhighly enriched mature osteoclasts (>95% of purity).Dexamethasone (Dex, 10^-10–10^-7 M) inhibited resorption pit formation on a dentine slice by the unfractionated bone cells in a dose- and time-dependent manner.However, Dex had no effect on the bone-resorbing activity of the isolated mature osteoclasts. When the isolated osteoclastswere co-cultured with rabbit osteoblastic cells, the osteoclastic bone resorption decreased in response to Dex,dependent on the number of osteoblastic cells. Like the effecton the bone resorption, Dex induced osteoclast apoptosis in cultures of the unfractionated bone cells, whereas it did not promote the apoptosis of the isolated osteoclasts. An inhibitorof caspases, Z-Asp-CH2-DCB attenuated both the inhibitory effecton osteoclastic bone resorption and the stimulatory effect onthe osteoclast apoptosis. In addition, the osteoblastic cellswere required for the osteoclast apoptosis induced by Dex. These findings indicate that the main target cells of GCs arenon-osteoclastic cells such as osteoblasts and that GCsindirectly inhibit bone resorption by inducing apoptosis ofthe mature osteoclasts through the action of non-osteoclasticcells. This study expands our knowledge about the multifunctional roles of GCs in bone metabolism.     

The Rho GTPase Wrch1 regulates osteoclast precursor adhesion and migration

An excess of osteoclastic bone resorption relative to osteoblastic bone formation results in progressive bone loss, characteristic of osteoporosis. Understanding the mechanisms of osteoclast differentiation is essential to develop novel therapeutic approaches to prevent and treat osteoporosis. We showed previously that Wrch1/RhoU is the only RhoGTPase whose expression is induced by RANKL during osteoclastogenesis. It associates with podosomes and the suppression of Wrch1 in osteoclast precursors leads to defective multinucleated cell formation. Here we further explore the functions of this RhoGTPase in osteoclasts, using RAW264.7 cells and bone marrow macrophages as osteoclast precursors. Suppression of Wrch1 did not prevent induction of classical osteoclastic markers such as NFATc1, Src, TRAP (Tartrate-Resistant Acid Phosphatase) or cathepsin K. ATP6v0d2 and DC-STAMP, which are essential for fusion, were also expressed normally. Similar to the effect of RANKL, we observed that Wrch1 expression increased osteoclast precursor aggregation and reduced their adhesion onto vitronectin but not onto fibronectin. We further found that Wrch1 could bind integrin ß3 cytoplasmic domain and interfered with adhesion-induced Pyk2 and paxillin phosphorylation. Wrch1 also acted as an inhibitor of M-CSF-induced prefusion osteoclast migration. In mature osteoclasts, high Wrch1 activity inhibited podosome belt formation. Nevertheless, it had no effect on mineralized matrix resorption. Our observations suggest that during osteoclastogenesis, Wrch1 potentially acts through the modulation of αvß3 signaling to regulate osteoclast precursor adhesion and migration and allow fusion. As an essential actor of osteoclast differentiation, the atypical RhoGTPase Wrch1/RhoU could be an interesting target for the development of novel antiresorptive drugs.     

Extracellular acidosis accelerates bone resorption by enhancing osteoclast survival, adhesion, and migration

Acidic extracellular pH promotes osteoporotic bone loss by osteoclast activation. However, the change of osteoclastic cell behavior in acidosis-stimulated bone resorption process is unknown. We found that lowering extracellular pH induced an increase in the survival, adhesion, and migration of mature osteoclasts with a full actin ring, leading to enhanced pit formation on dentine slices. Acidosis upregulated osteopontin, which is an Arg-Gly-Asp (RGD) motif-containing matrix protein secreted from osteoclasts and acts as a common modulator for their survival, adhesion, and migration. A synthetic RGD peptide treatment blocked acidosis-induced osteoclast adhesion and migration, likely by competing with the RGD motif-containing extracellular matrix proteins for cell surface integrin binding. We finally observed that acidosis was associated with activation of osteoclast survival/adhesion/migration-related Pyk2, Cbl-b, and Src signals. Collectively, the findings indicate that extracellular acidosis stimulates bone resorption by extending osteoclast survival and facilitating osteoclast adhesion and migration.     

Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.     

Hedgehog signaling induced by breast cancer cells promotes osteoclastogenesis and osteolysis

Bone integrity is maintained by a dynamic equilibrium between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. Osteolytic lesions are a painful consequence of metastasis of breast cancer cells to bone in an overwhelming majority of breast cancer patients. Factors secreted by breast cancer cells propel a cascade of events that trigger osteoclastogenesis and elevated bone resorption. In the present study, we show that the Hedgehog (Hh) ligands secreted by breast cancer cells promote osteoclast differentiation and potentiate the activity of mature osteoclasts. Paracrine Hh signaling induced by breast cancer cells mediates a detrimental chain of events by the up-regulation of osteopontin (OPN), which in turn enhances osteoclastic activity by up-regulating cathepsin K and MMP9. Hh signaling is essential for osteoclasts because blocking the Hh pathway using the pharmacological Hh inhibitor, cyclopamine, results in an overall decrease in osteoclastogenesis and resorptive activity. Our studies suggest that inhibiting Hh signaling interferes with the ability of pre-osteoclasts to respond to the stimulatory effects of the breast cancer cells, indicating that Hh signaling is vital to osteoclast activity.     

Distinct osteoclast precursors in the bone marrow and extramedullary organs characterized by responsiveness to Toll-like receptor ligands and TNF-alpha

Osteoclasts are derived from hemopoietic stem cells and play critical roles in bone resorption and remodeling. Multinucleated osteoclasts are attached tightly to bone matrix, whereas precursor cells with the potential to differentiate into osteoclasts in culture are widely distributed. In this study, we assessed the characteristics of osteoclast precursors in bone marrow (BM) and in extramedullary organs as indicated by their responsiveness to ligands for Toll-like receptors (TLRs) and to TNF-alpha. Development of osteoclasts from precursor cells in the BM was inhibited by CpG oligonucleotides, a ligand for TLR9, but not by LPS, a ligand for TLR4. BM osteoclasts were induced by TNF-alpha as well as receptor activator of NF-kappaB ligand in the presence of M-CSF. Splenic osteoclast precursors, even in osteoclast-deficient osteopetrotic mice, differentiated into mature osteoclasts following exposure to TNF-alpha or receptor activator of NF-kappaB ligand. However, splenic osteoclastogenesis was inhibited by both LPS and CpG. Osteoclastogenesis from peritoneal precursors was inhibited by not only these TLR ligands but also TNF-alpha. The effects of peptidoglycan, a ligand for TLR2, were similar to those of LPS. BM cells precultured with M-CSF were characterized with intermediate characteristics between those of splenic and peritoneal cavity precursors. Taken together, these findings demonstrate that osteoclast precursors are not identical in the tissues examined. To address the question of why mature osteoclasts occur only in association with bone, we may characterize not only the microenvironment for osteoclastogenesis, but also the osteoclast precursor itself in intramedullary and extramedullary tissues.     

Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.     

CELLULAR BIOLOGY OF BONE RESORPTION

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged.
1. The resorption of bone is the result of successive steps that can be regulated individually.
2. Osteoclast progenitors are formed in bone marrow. This is followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone.
3. The exact pathways of differentiation of the osteoclast progenators to mature osteoclasts are debatable, but there is clear evidence that stromal cells support osteoclast generation.
4. Osteoclasts are activated following contact with mineralized bone. This appears to be controlled by osteoblasts that expose mineral to osteoclasts and/or release a factor that activates these cells.
5. Activated osteoclasts dissolve the bone mineral and digest the organic matter of bone by the action of agents secreted in the segregated microcompartments underlying their ruffled borders. The mineral is solubilized by protons generated from CO, by carbonic anhydrase and secreted by an ATP-driven vacuolar H⁺-K⁺-ATPase located at the ruffled border. The organic matrix of the bone is removed by acid proteinases, particularly cysteine-proteinases that are secreted together with other lysosomal enzymes in the acid environment of the resorption zone.
6. Osteoclastic bone resorption is directly regulated by a polypeptide hormone, calcitonin (CT), and locally, by ionized calcium (Ca²⁺) generated as a result of osteoclastic bone resorption.
7. There is new evidence that osteoclast activity may also be influenced by the endothelial cells via generation of products including PG, NO and endothelin.     

Extracellular glutamate alters mature osteoclast and osteoblast functions

Glutamatergic intercellular communication is involved in many aspects of metabolic homeostasis in normal bone. In bone metastasis, the balance between bone formation and degradation is disrupted. Although the responsible mechanisms are not clear, we have previously identified that cancer cell lines used in bone tumour models secrete glutamate, suggesting that tumour-derived glutamate may disrupt sensitive signalling systems in bone. This study examines the role of glutamate in mature osteoclastic bone resorption, osteoblast differentiation, and bone nodule formation. Glutamate was found to have no effect on the survival or activity of mature osteoclasts, although glutamate transporter inhibition and receptor blockade increased the number of bone resorption pits. Furthermore, transporter inhibition increased the area of resorbed bone while significantly decreasing the number of osteoclasts. Alkaline phosphatase activity and extracellular matrix mineralization were used as measurements of osteoblast differentiation. Glutamate significantly increased osteoblast differentiation and mineralization, but transport inhibitors had no effect. These studies support earlier findings suggesting that glutamate may be more important for osteoclastogenesis than for osteoclast proliferation or functions. Since glutamate is capable of changing the differentiation and activities of both osteoclast and osteoblast cell types in bone, it is reasonable to postulate that tumour-derived glutamate may impact bone homeostasis in bone metastasis.     

Prostaglandin E2 receptors EP2 and EP4 are down-regulated during differentiation of mouse osteoclasts from their precursors

Prostaglandin E2 (PGE2) has been proposed to be a potent stimulator of bone resorption. However, PGE2 itself has been shown to directly inhibit bone-resorbing activity of osteoclasts. We examined the role of PGE2 in the function of mouse osteoclasts formed in vitro. Bone marrow macrophage osteoclast precursors expressed PGE2 receptors EP1, EP2, EP3beta, and EP4, and the expression of EP2 and EP4 was down-regulated during osteoclastic differentiation induced by receptor activator of NF-kappaB ligand and macrophage colony-stimulating factor. In contrast, functional EP1 was continuously expressed in mature osteoclasts. PGE2 as well as calcitonin caused intracellular Ca2+ influx in osteoclasts. However, PGE2 and 17-phenyltrinol-PGE2 (an EP1 agonist) failed to inhibit actin-ring formation and pit formation by osteoclasts cultured on dentine slices. When EP4 was expressed in osteoclasts using an adenovirus carrying EP4 cDNA, both actin-ring and pit-forming activities of osteoclasts were inhibited in an infectious unit-dependent manner. Treatment of EP4-expressing osteoclasts with PGE2 further inhibited their actin-ring and pit-forming activities. Such inhibitory effects of EP4-mediated signals on osteoclast function are similar to those that are calcitonin receptor-mediated. Thus, osteoclast precursors down-regulate their own EP2 and EP4 levels during their differentiation into osteoclasts to escape inhibitory effects of PGE2 on bone resorption.     

17beta-estradiol downregulates beta3-integrin expression in differentiating and mature human osteoclasts

The increased bone resorption observed after estrogen withdrawal is responsible for bone loss and may lead to osteoporosis. The mechanism by which estradiol inhibits bone resorption is known to involve decreased osteoclastogenesis, however, the effect on osteoclast adhesion remains unclear. We examined the in vitro effect of estradiol and raloxifene on human osteoclast differentiation and function. Human peripheral blood mononuclear cells were cultured with M-CSF/RANK-L for 18 days, and we evaluated bone resorption, the expression of the protein and mRNA of the integrins, c-jun and c-fos in the presence or absence of estradiol. In this human model, beta3-integrin expression increased at the mRNA and protein levels during osteoclast differentiation, whereas that of beta5-integrin did not. We found that estradiol and raloxifene directly inhibited bone resorption on bone slices by 50%, and decreased the expression of beta3-integrin mRNA (60%) and protein (20%) in a time-dependent manner. Moreover, the mRNAs of c-fos and c-jun were both diminished by estradiol and raloxifene, particularly in early osteoclasts, but also to a lesser extent in mature cells. These findings suggest that the direct inhibitory action of estradiol on bone resorption may affect human osteoclast differentiation through downregulation of c-fos and c-jun and adhesion through modulation of beta3-integrin.     

Glycosaminoglycans inhibit the adherence and the spreading of osteoclasts and their precursors: role in osteoclastogenesis and bone resorption

The bone microenvironment (e.g. glycosaminoglycans (GAGs), growth factors) plays a major role in bone resorption, especially in the formation of osteoclasts which differentiate from the hematopoietic lineage in the presence of RANKL. Previous studies revealed that GAGs may influence osteoclastogenesis, but data are very controversial, some studies showing an inhibitory effect of GAGs on osteoclastic differentiation whereas others demonstrated a stimulatory effect. To clarify their activities, we investigated the effect of 5 families of GAGs in three different models of human/mouse osteoclastogenesis. The present data revealed that heparin inhibited osteoclastogenesis in these three models, which was confirmed by a decrease in mRNA expression of osteoclastic markers and by an inhibition of the bone resorption capacity. We also demonstrated in RAW 264.7 cells that other families of GAGs different from heparin inhibited RANKL-induced osteoclastogenesis, and that this inhibition was dependent on the length and the level of sulfation of GAGs. In the present work, heparin did not bind to RANKL and did not modulate RANKL signaling. Heparin acted at 2 distinct steps of osteoclastogenesis from human CD14(+) cells: first, heparin strongly decreased the adherence of osteoclast precursors, and secondly inhibited osteoclasts to spread and to be active. Furthermore, the second action of heparin was reversible as the removal of heparin at the end of the culture time allowed the condensed cells to spread out and showed the formation of morphological active osteoclasts. The present work clearly evidences that GAGs inhibit osteoclastogenesis in vitro and strengthens the therapeutic interest of defined GAGs in osteolytic diseases.     

Effects of interleukin 3 and of granulocyte-macrophage and macrophage colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue

The effects of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 3 (IL3) on osteoclast formation were tested by incubation of murine hemopoietic cells on plastic coverslips and bone slices with GM-CSF, M-CSF, or IL3, with or without 1,25(OH)2 vitamin D3 (1,25(OH)2D3). Osteoclastic differentiation was detected after incubation by scanning electron microscopical examination of bone slices for evidence of osteoclastic excavations, and by autoradiographic assessment of cells for 1,25(OH)2D3-calcitonin (CT) binding. The differentiation of CT-receptor-positive cells preceded bone resorption, but the number that developed correlated with the extent of bone resorption (r = 0.88). M-CSF and GM-CSF substantially reduced bone resorption and CT-receptor-positive cell formation. The degree of inhibition of bone resorption could not be attributed to effects on the function of mature cells, since M-CSF inhibits resorption by such cells only by 50%, and GM-CSF has no effect. GM-CSF inhibited the development of mature function (bone resorption) to a greater extent than it inhibited CT-receptor-positive cell formation. Since CT-receptor expression antedated resorptive function, this suggests that GM-CSF resulted in the formation of reduced numbers of relatively immature osteoclasts. This suggests that it may exert a restraining effect on the maturation of cells undergoing osteoclastic differentiation in response to 1,25(OH)2D3. Conversely, IL3, which also has no effect on mature osteoclasts, by itself induced CT-receptor expression but not bone resorption; in combination with 1,25(OH)2D3 it induced a threefold increase in bone resorption and CT-receptor-positive cells compared with cultures incubated with 1,25(OH)2D3 alone. IL3 did not induce CT-receptors in peritoneal macrophages, blood monocytes, or J 774 cells. The results suggest that IL3 induces only partial maturation of osteoclasts, which is augmented or completed by additional factors such as 1,25(OH)2D3.     

Gallium inhibits bone resorption by a direct effect on osteoclasts

Gallium nitrate has been used clinically to treat cancer-related hypercalcemia. It has been suggested that gallium may reduce calcium release from bone by inhibiting bone resorption, but the mechanism(s) involved remain to be elucidated. Therefore, we have examined the effect of gallium on bone resorption in vitro using osteoclasts isolated from neonatal rat long bones cultured on slices of cortical bone. Gallium nitrate (0.01–100 μg/ml) produced a concentration-dependent inhibition of bone resorption. Morphological studies showed that even (100μg/ml) gallium nitrate induced no light microscopical change in osteoclast morphology and did not affect their survival on bone slices. Pretreatment of bone slices with gallium nitrate (100μg/ml for 18 h), followed by extensive washing also inhibited subsequent osteoclastic bone resorption. These results suggest that gallium can be adsorbed onto the calcified surface of bone and inhibit osteoclastic bone resorption.     

NMDA glutamate receptors are expressed by osteoclast precursors and involved in the regulation of osteoclastogenesis

We previously identified functional N-methyl-D-aspartate (NMDA) glutamate receptors in mature osteoclasts and demonstrated that they are involved in bone resorption in vitro. In the present work, we studied the expression of NMDA receptors (NMDAR) by osteoclast precursors and their role in osteoclastogenesis using two in vitro models, the murine myelomonocytic RAW 264.7 cell line and mouse bone marrow cells, both of which differentiate into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and Rank ligand (RankL). Using RT-PCR analysis with specific probes, we showed that RAW 264.7 cells and mouse bone marrow cells express mRNA of NMDAR subunits NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) A, B, and D. These subunits are expressed all along the differentiation sequence from undifferentiated precursors to mature resorbing osteoclasts. Semi-quantitative PCR analysis showed no regulation of the expression of these subunits during the differentiation process. Two specific non competitive antagonists of NMDAR, MK801 and DEP, dose-dependently inhibited osteoclast formation in both models, indicating that osteoclastogenesis requires the activation of NMDAR expressed by osteoclast precursors. MK801 had no effect when added only during the first 2 days of culture, suggesting that NMDAR are rather involved in the late stages of osteoclast formation. Finally, we demonstrated using Western-blotting and immunofluorescence that activation of NMDAR in RAW 264.7 cells by specific agonists induces nuclear translocation of NF-kappa B, a factor required for osteoclast formation. Altogether, our results indicate that osteoclast precursors express NMDAR that are involved in the osteoclast differentiation process through activation of the NF-kappa B pathway.     

Lymphocytes and the Dap12 adaptor are key regulators of osteoclast activation associated with gonadal failure

Bone resorption by osteoclasts is necessary to maintain bone homeostasis. Osteoclast differentiation from hematopoietic progenitors and their activation depend on M-CSF and RANKL, but also requires co-stimulatory signals acting through receptors associated with DAP12 and FcRgamma adaptors. Dap12 mutant mice (KDelta75) are osteopetrotic due to inactive osteoclasts but, surprisingly, these mice are more sensitive than WT mice to bone loss following an ovariectomy. Because estrogen withdrawal is known to disturb bone mass, at least in part, through lymphocyte interaction, we looked at the role of mature lymphocytes on osteoclastogenesis and bone mass in the absence of functional DAP12. Lymphocytes were found to stimulate an early osteoclast differentiation response from Dap12-deficient progenitors in vitro. In vivo, Rag1-/- mice lacking mature lymphocytes did not exhibit any bone phenotype, but lost their bone mass after ovariectomy like KDelta75 mice. KDelta75;Rag1-/- double mutant female mice exhibited a more severe osteopetrosis than Dap12-deficient animals but lost their bone mass after ovariectomy, like single mutants. These results suggest that both DAP12 and mature lymphocytes act synergistically to maintain bone mass under physiological conditions, while playing similar but not synergistic co-stimulatory roles in protecting bone loss after gonadal failure. Thus, our data support a role for lymphocytes during osteoclast differentiation and suggest that they may function as accessory cells when regular osteoclast function is compromised.